RNA-directed RNA polymerases are well known, as are compatible templates that they replicate. Q.beta. replicase, an RNA-directed RNA polymerase, is known to be compatible with, i.e., to replicate, certain templates exponentially, including MDV-1 RNA, a small, naturally occurring template, and RQ135 RNA. We are aware that Q.beta. replicase is compatible with and exponentially amplifies recombinant RNAs which comprise a messenger RNA (an mRNA) sequence embedded within the sequence of MDV-1 RNA. We are also aware that for short periods of time the amplified product can be used as a template for the synthesis of its encoded protein in a batch cell-free translation system.
Teachings in the art indicate that the complex and stable secondary and tertiary structures present in full-length recombinant mRNA limit the access of ribosomes to the protein initiation site. Teachings in the art indicate that exponential amplification, which will generate many copies of both plus (+ or sense) and minus (- or antisense) strands of recombinant mRNA, can lead to the hybridization of plus and minus strands to each other over the long term, rendering the plus strands useless for protein synthesis through translation by ribosomes unless the hybridized strands are melted apart prior to use. "Protein" as used herein means the polypeptide synthesized by a ribosome in response to the mRNA that encodes it. Exponential amplification generates both plus and minus strands. Teachings in the art also indicate that replication over a prolonged period, that is, the copying of copies, leads to an accumulation of mutated strands not suitable for synthesis of a desired protein. For these reasons the art suggests that the amplification of recombinant mRNAs by Q.beta. replicase for use as templates for cell-free translation would not be satisfactory for the production of protein. In particular, one would anticipate that amplification time would have to be kept short (less than one hour), which would be very limiting and not advantageous compared to uncoupled or coupled transcription-translation methods. Furthermore, the art suggests that replication would not be useful in coupled methods, such as transcription-translation, because of the need to melt apart hybridized strands of replicated recombinant mRNA.
It is an objective of this invention to utilize the ability of RNA-directed RNA polymerases such as Q.beta. replicase to generate large amounts of the recombinant mRNAs to generate templates for protein synthesis while overcoming the three problems, discussed above, taught by the art.